Blockade of endogenous neurotrophic factors prevents the androgenic rescue of rat spinal motoneurons.

Target-derived neurotrophic factors are assumed to regulate motoneuron cell death during development but remain unspecified. Motoneuron cell death in the spinal nucleus of the bulbocavernosus (SNB) of rats extends postnatally and is controlled by androgens. We exploited these features of the SNB system to identify endogenously produced trophic factors regulating motoneuron survival. Newborn female rat pups were treated with the androgen, testosterone propionate, or the oil vehicle alone. In addition, females received trophic factor antagonists delivered either into the perineum (the site of SNB target muscles) or systemically. Fusion molecules that bind and sequester the neurotrophins (trkA-IgG, trkB-IgG, and trkC-IgG) were used to block activation of neurotrophin receptors, and AADH-CNTF was used to antagonize signaling through the ciliary neurotrophic factor receptor-alpha (CNTFRalpha). An acute blockade of trkB, trkC, or CNTFRalpha prevented the androgenic sparing of SNB motoneurons when antagonists were delivered to the perineum. Trophic factor antagonists did not significantly reduce SNB motoneuron number when higher doses were injected systemically. These findings demonstrate a requirement for specific, endogenously produced trophic factors in the androgenic rescue of SNB motoneurons and further suggest that trophic factor interactions at the perineum play a crucial role in masculinization of this neural system.

Additive effects of ciliary neurotrophic factor and testosterone on motoneuron survival; differential effects on motoneuron size and muscle morphology.

Testosterone and ciliary neurotrophic factor (CNTF) each enhance motoneuron survival in the spinal nucleus of the bulbocavernosus (SNB) of newborn rats. Here we directly compared the effects of CNTF and testosterone, alone and in combination, on SNB motoneuron number, SNB cell size, and morphology of the levator ani (LA) target muscle. Female rat pups were treated daily from postnatal day 1 through 6 (P1-P6) with recombinant human CNTF (hCNTF), testosterone propionate (TP), both hCNTF and TP, or neither. Effects of treatment were assessed on P7. TP and hCNTF each increased the number of SNB motoneurons and did so to a similar degree. Females treated with both hCNTF and TP had significantly more SNB cells than those receiving either hCNTF or TP alone. TP administered from P1 to P6 also increased SNB motoneuron size on P7. In contrast, hCNTF alone did not significantly affect SNB cell size, and hCNTF in combination with TP antagonized the effect of TP on motoneuron size. TP also increased LA muscle fiber number and LA fiber size, whereas hCNTF did not significantly influence LA muscle morphology in this study. Immunohistochemistry established that virtually all SNB motoneurons of both males and females express the CNTF alpha receptor (CNTFRalpha) between embryonic day 20 and postnatal day 6. Thus, effects of TP and hCNTF on SNB motoneuron survival were additive, and increases in motoneuron survival were dissociated from changes in target muscle morphology in hCNTF-treated animals. SNB motoneurons express CNTFRalpha perinatally and are therefore potential direct sites of hCNTF action.

Retinal afferents to the dorsal raphe nucleus in rats and Mongolian gerbils.

A direct pathway from the retina to the dorsal raphe nucleus (DRN) has been demonstrated in both albino rats and Mongolian gerbils. Following intraocular injection of cholera toxin subunit B (CTB), a diffuse stream of CTB-positive, fine-caliber optic axons emerged from the optic tract at the level of the pretectum/anterior mesencephalon. In gerbils, CTB-positive axons descended ventromedially into the periaqueductal gray, moving caudally and arborizing extensively throughout the DRN. In rats, the retinal-DRN projection comprised fewer, but larger caliber, axons, which arborized in a relatively restricted region of the lateral and ventral DRN. Following injection of CTB into the lateral DRN, retrogradely labeled ganglion cells (GCs) were observed in whole-mount retinas of both species. In gerbils, CTB-positive GCs were distributed over the entire retina, and a nearest-neighbor analysis of CTB-positive GCs showed significant regularity (nonrandomness) in their distribution. The overall distribution of gerbil GC soma diameters ranged from 8 to 22 micrometer and was skewed slightly towards the larger soma diameters. Based on an adaptive mixtures model statistical analysis, two Gaussian distributions appeared to comprise the total GC distribution, with mean soma diameters of 13 (SEM +/-1.7) micrometer, and 17 (SEM +/-1.5) micrometer, respectively. In rats, many fewer CTB-positive GCs were labeled following CTB injections into the lateral DRN, and nearly all occurred in the inferior retina. The total distribution of rat GC soma diameters was similar to that in gerbils and also was skewed towards the larger soma diameters. Major differences observed in the extent and configuration of the retinal-DRN pathway may be related to the diurnal/crepuscular vs. nocturnal habits of these two species.

Ciliary neurotrophic factor receptor alpha in spinal motoneurons is regulated by gonadal hormones.

Ciliary neurotrophic factor receptor alpha (CNTFRalpha) is the ligand-binding component of the CNTF receptor. CNTFRalpha expression is essential for the normal development of spinal motoneurons and is required for the development of a sex difference in motoneuron number in androgen-sensitive perineal motoneurons. We used immunocytochemistry to examine the expression and hormone regulation of CNTFRalpha protein in the spinal nucleus of the bulbocavernosus (SNB), dorsolateral nucleus and retrodorsolateral nucleus of the lower lumbar spinal cord of adult rats. CNTFRalpha immunoreactivity (CNTFRalpha-IR) was observed in the somata and dendrites of virtually all motoneurons. In all three motor pools, the intensity of motoneuron soma labeling was greatest among gonadally intact males and was reduced in females and gonadectomized males. The density of CNTFRalpha-IR in neuropil also tended to be highest in intact males. Short-term (2 d) testosterone propionate treatment reversed the decline in the density of soma labeling in the SNB of castrated males but did not reverse any other effects of castration. Long-term hormone treatment, achieved by implanting males with testosterone capsules at the time of gonadectomy, prevented the decline in soma labeling in all motor pools and partially prevented the decline in neuropil label caused by castration. We conclude that expression of CNTFRalpha protein is androgen-regulated in spinal motoneurons.

Sexual dimorphism in the spinal cord is absent in mice lacking the ciliary neurotrophic factor receptor.

Ciliary neurotrophic factor (CNTF) has potent survival-promoting effects on motoneurons in vitro and in vivo. We examined knockout mice with null mutations of the gene for either CNTF itself or the alpha-subunit of the CNTF receptor (CNTFRalpha) to assess whether CNTF and/or its receptors are involved in the development of a sexually dimorphic neuromuscular system. Male rodents have many more motoneurons in the spinal nucleus of the bulbocavernosus (SNB) than do females. This sex difference is caused by hormone-regulated death of SNB motoneurons and their target muscles. Sexual dimorphism of SNB motoneuron number developed completely normally in CNTF knockout (CNTF -/-) mice. In contrast, a sex difference in the SNB was absent in CNTFRalpha -/- animals: male mice lacking a functional CNTF alpha-receptor had fewer than half as many SNB motoneurons than did wild-type males and no more than did their female counterparts. Size of the bulbocavernosus and levator ani muscles, the main targets of SNB motoneurons, was not affected in either CNTF or CNTFRalpha knockout males. These observations suggest that signaling through the CNTF receptor is involved in sexually dimorphic development of SNB motoneuron number and that target muscle survival per se is not sufficient to ensure motoneuron survival in this system. In addition, our observations are consistent with the suggestion that CNTF itself is not the only endogenous ligand for the CNTF receptor. A second, as yet unknown, ligand may be important for neural development, including sexually dimorphic motoneuron development.

Short- and long-term effects of ciliary neurotrophic factor on androgen-sensitive motoneurons in the lumbar spinal cord.

Motoneuron death in the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN) of the lumbar spinal cord is androgen regulated. As a result, many more SNB and DLN motoneurons die in perinatal female rats than in males, whereas treatment of newborn females with androgen results in a permanent sparing of the motoneurons and their target muscles. We previously observed that a neurotrophic molecule, ciliary neurotrophic factor (CNTF), also arrests the death of SNB motoneurons and their target musculature, at least in the short term. The present study compares the short- and long-term consequences of perinatal CNTF treatment on motoneuron number in the SNB, the DLN, and the retrodorsolateral nucleus (RDLN), a motor pool in the lower lumbar cord that does not exhibit hormone-regulated cell death. Female pups were treated with CNTF or vehicle alone from embryonic day 22 through postnatal day 6 (P6). Motoneuron number in the each nucleus was then determined immediately after treatment on P7, or 10 weeks later (P77). CNTF treatment significantly elevated motoneuron number in the SNB and DLN on P7; the volume of SNB target muscles on P7 was also greater in the CNTF-treated group. These effects were transient, however, as motoneuron number and ratings of muscle size were not different in CNTF-and vehicle-treated females on P77. Perinatal CNTF treatment did not alter cell number in the RDLN at either age. The finding that effects of CNTF on SNB and DLN motoneuron number are short lived contrasts with the permanent effects of early androgen treatment, and has implications for molecular models of the actions of androgen and neurotrophic factors on the developing spinal cord.

Experimental light damage increases lipofuscin in the retinal pigment epithelium of Japanese quail (Coturnix coturnix japonica).

Lipofuscin accumulation in the retinal pigment epithelium (RPE) of Japanese quail was investigated in normal and light-damaged animals at 4 months and 12 months of age following a single, 18-hr light-damaging exposure (3000-3200 lx) evaluated 6 weeks postexposure. Quail in a second experimental group light-damaged at 4 months of age were exposed to a second, identical light-damaging exposure at 12 months of age. RPE lipofuscin was quantitatively assessed in three ways: (1) total lipofuscin, expressed as a percentage of the basal RPE cell area, (2) mean number of lipofuscin granules per RPE cell, and (3) mean area of lipofuscin granules. At 12 months of age, control female RPE cells showed substantially more lipofuscin than did males. Animals that received a single damaging light-exposure showed no significant differences in lipofuscin at either age when compared with controls, and none were observed between males and females. However, in the double light-exposure condition, both total lipofuscin and the number of lipofuscin granules were significantly higher in females compared to controls and also, when compared with animals in the single light-exposure condition. Mean lipofuscin granule size decreased in females in the double light-exposure condition when compared with control values. Histopathological evaluation of photoreceptor outer segments and outer nuclear layer indicated that double-exposed, 12-month-old females showed the most severe effects, including a substantial decrease (70%) in rod photoreceptor densities. However, only a small change was observed in cone densities for either sex (12-15%) in this condition. Overall, a strong, negative correlation (r = -0.78) was obtained between total lipofuscin and average rod density across age, sex, control and experimental conditions. A somewhat greater correlation (r = -0.82) was obtained for females, alone. These results suggest that rods, when severely damaged by light exposure, contribute to increased RPE lipofuscin. Age, sex and light-exposure history appear to be critical variables that can influence both the amount of lipofuscin present in RPE cells as well as the relative vulnerability of rod and cone photoreceptors to cumulative light-exposure damage. These results have implications for the development of a variety of age-related changes and diseases of the outer retina, including age-related macular degeneration.

Metabolic correlates of optokinetic stimulation in the central visual system of the frog, Rana pipiens.

The [14C]2-deoxyglucose (2-DG) method was used to identify those structures in the central visual system of Rana pipiens showing increased metabolic activity during binocular and monocular optokinetic stimulation at two pattern velocities (2 deg/sec and 9 deg/sec). Analysis of autoradiograms made with computer-assisted microdensitometry and pseudocolor image-enhancement techniques revealed that the greatest uptake of 2-DG occurred in the pretectal region, which included the large-celled nucleus lentiformis mesencephali (nLM), nucleus pretectalis, and pretectal gray. Both temporal-to-nasal (T-N) and nasal to temporal (N-T) directions of pattern motion were correlated with high levels of 2-DG uptake. However, the nucleus of the basal optic root (nBOR) showed the greatest uptake of 2-DG for the N-T direction, which evokes little or no optokinetic (OKN) response in this species. These results suggest that the major efferent projection from nBOR to nLM may exert its greatest effect upon the ipsilateral pretectum during N-T stimulation. Other regions showing substantial 2-DG uptake included a large region of the ventral thalamus, for all stimulus conditions tested, including those cell groups associated with the retinorecipient neuropil, corpus geniculatum. The pattern of uptake was less closely associated with specific parameters of optokinetic stimulation than was observed in the pretectum and accessory optic nucleus. Like nBOR, the auricular lobe of the cerebellum also showed greatest uptake of 2-DG for the N-T, monocular stimulus condition. The neural circuitry that underlies OKN and its directional asymmetry during monocular stimulation appears to involve a number of structures whose functional interrelationships are yet to be described.

Aging and sex-related changes in the outer retina of Japanese quail.

Age-related changes in the outer retina of Japanese quail 3 months to 3 years of age have been assessed with light and electron microscopy. A major difference was observed between males and females in the accumulation of lipofuscin in retinal pigment epithelial (RPE) cells. Females showed greater densities of lipofuscin granules, larger granules, and more lipofuscin per RPE cell than did males of comparable ages. In addition, a small but significant decrease (14-16%) in photoreceptor nuclear densities occurred in the temporal retina of both 1-year females and 3-year males. An overall correlation of -.77 between photoreceptor densities and amount of lipofuscin was observed, with a correlation of -.88 for females, alone. No male/female differences were observed with regard to age-related changes in height of RPE cells. Major differences in calcium metabolism and demand associated with egg-laying in females may underlie the observed sex-related differences that occur in the outer retina of this relatively short-lived, domestic species.

Neurophysiological investigation of the pretectal nucleus lentiformis mesencephali in Rana pipiens.

Previous studies have demonstrated that the large-celled, optic pretectal nucleus lentiformis mesencephali (nLM) is essential for horizontal optokinetic nystagmus, yet little is known about its neurophysiology. In the present study, single-unit analysis of nLM utilized a large-field, patterned stimulus presented for 8 directions and 3 velocities of movement. All units localized in nLM were spontaneously active, motion sensitive, with response profiles that ranged from strongly directional and narrowly tuned to asymmetric and broadly tuned. Only about one-third of the units could be classified as directional, and no response bias for horizontal or temporal-to-nasal motion was observed. The majority of directional units showed greatest responsiveness at the lowest stimulus velocity, while the reverse occurred for many broadly tuned units. These low-velocity, highly directional units may be comparable to the 'retinal slip' neurons recently described in the large-celled pretectal nucleus of mammals. Directional information in the nLM of Rana pipiens thus appears to be represented in the activity of a large population of motion-sensitive units which includes both narrowly and broadly tuned individual response profiles. These results are consistent with the population-coding hypothesis recently advanced to account for directional coding in other sensorimotor systems, including primate motor cortex and superior colliculus.

Anuran accessory optic system: evidence for a dual organization.

The accessory optic system of Rana pipiens consists of lateral and medial fascicles of the basal optic root (BOR1, BORm) and a single terminal nucleus, nBOR. The present study provides new evidence that these two fascicles differ not only in their trajectories but in fiber spectra and innervation patterns as well. BOR1 contains a substantially higher percentage of large, myelinated axons than does BORm, and the mean diameter of axons in BOR1 is greater than that of BORm. BOR1 innervates the entire terminal field of nBOR, while BORm innervates only the central and mediodorsal portions of nBOR. The ventrolateral portion of nBOR is uniquely innervated by BOR1 and contains several types of neurons not found in the central and medial regions of nBOR which are innervated by both fascicles. Cytoarchitectural analysis of nBOR with Golgi techniques has revealed a number of similarities between the anuran nBOR and the mammalian medial terminal nucleus (MTN) with regard to cellular morphology, dendritic geometry, and retinofugal arborization patterns. In frog, nBOR appears comparable to the ventral subdivision of the mammalian MTN, while the peri-nBOR region, which contains neurons postsynaptic to nBOR, may represent a more primordial version of the mammalian dorsal MTN.

Enhanced integumental and ocular amelanosis following the termination of cyclosporine administration.

The Smyth delayed amelanotic line of chickens display symptoms commonly associated with human vitiligo. Administration of the immunosuppressive compound, cyclosporine, significantly delayed the mean age of onset and incidence of integumental pigment losses in this mutant line of vitiliginous chickens. Associated ocular pathology was also less severe in treated chicks. Termination of cyclosporine administration resulted in enhanced integumental and choroidal amelanosis, choroidal inflammation, and chorioretinal damage beyond that observed in nontreated controls. These results suggest that withdrawal of cyclosporine in treatment of this spontaneous autoimmune disease may exacerbate associated symptoms.

Effects of cyclosporine in spontaneous, posterior uveitis.

Cyclosporin A (CsA) was administered to chicks of the Smyth delayed-amelanotic (SDA) line from day of hatch to 4, 8 or 12 weeks of age. Animals were evaluated at 8, 12, 16 or 20 weeks with regard to major features of the SDA-line disorder, including extent of feather and choroidal amelanosis, choroidal inflammation, and histopathology of the retinal pigment epithelium and outer retina. A suppression and delay in the onset of both amelanosis and ocular histopathology occurred during CsA administration. However, a rebound enhancement of symptoms occurred 4-8 weeks after withdrawal of CsA that was closely associated with the duration of CsA treatment. These results indicate that CSA may yield therapeutic effects during the period of treatment, but its withdrawal may lead to more severe symptoms that would have occurred without treatment.

Retinal pigment epithelial correlates of avian retinal degeneration: electron microscopic analysis.

The delayed amelanotic (DAM) strain of domestic chicken is characterized by an early, developmental onset of choroidal inflammation and destruction of both feather and choroidal melanocytes. Secondarily, retinal pigment epithelial (RPE) cells in the peripapillary region develop abnormalities, and a series of progressive histopathological changes ensues which includes reduction and ultimate loss of RPE-melanin granules and RPE-cell atrophy. The earliest sign of RPE-cell abnormality is a dramatic alteration in the distribution of intracellular melanin granules. Apical processes also show a lessening of contact with photoreceptor outer segments, leading in more advanced stages to their retraction and development of retinal detachments. Other progressive alterations in RPE cells include disorganization and loss of basal infoldings; size reductions and density increases in both mitochondria and myeloid bodies from early to advanced stages; appearance of large macrophages in the subretinal space; Loss of intercellular junctional complexes; and progressive reduction in the density of melanin granules. These abnormalities appear to spread in a cell-by-cell, radial pattern, until widespread areas of the retina become severely pathologic and atrophic. The DAM chorioretinal disorder appears to show many of the histopathologic features which characterize experimentally induced uveitis and other ocular diseases which may result from hypersensitivity to, or autoimmune reaction against, pigments of the uveal tract.

Retinal degeneration in the delayed amelanotic (DAM) chicken: an electroretinographic study.

The retinal function of the delayed amelanotic (DAM) chicken that has progressive retinal degeneration, was investigated electroretinographically (ERG). Subjects showing a gradation of abnormalities of retinal morphology and impairment of visual behaviors were tested. Dark-adapted a-, b-, and c-wave responses to full-field stimuli were studied. In general, the more severe the anatomical abnormalities, the greater the compromise of sensitivity and amplitude of ERG responses. The DAM diseases appears to affect c-waves more than a-waves, and a-waves more than b-waves. Dark-adapted responses are affected more than light-adapted responses. In DAM's with moderately to severely impaired visual behaviors, ERG studies should permit analysis of the distal retinal pathophysiology caused by this disease.

Neural correlates of optokinetic nystagmus in the mesencephalon of Rana pipiens: a functional analysis.

The effects of lesions of the anuran mesencephalic retinal terminal fields on horizontal optokinetic nystagmus (OKN) were examined. Lesion sites which produced effects upon OKN responses were as follows: BOR, nBOR, peri-nBOR, the large-celled pretectal nucleus, and the dorsal tegmental gray and deep tectal layers. Transection of BOR generally resulted in an increase in saccadic frequently at the lower stimulus velocities. Lesions of nBOR produced a decrease in the frequency of both head and eye saccades in the middle to high range of stimulus velocities. The only lesions which totally abolished horizontal OKN were those located medical to nBOR, in the peri-nBOR region. Lesions of the large-celled pretectal nucleus and dorsal tegmental gray substantially reduced both head and eye saccades at all stimulus velocities. Small lesions in the deep tectal layers also depressed OKN frequency. These studies indicate that horizontal OKN may be mediated by a number of structures within the anuran mesencephalon.

The accessory optic system of Rana pipiens: neuroanatomical connections and intrinsic organization.

The accessory optic system of Rana pipiens was investigated by autoradiographic, horseradish peroxidase, and Golgi techniques, revealing a complexity of neuroanatomical organization previously unrecognized. Retinal afferents project to the nucleus of the basal optic root (nBOR) via a primary bundle and more diffuse, medial bundle of optic axons, both of which contain large- and small-diameter fibers. At least six types of retinal ganglion cell contribute to the basal optic root (BOR), including giant ganglion cells, two intermediate-sized ganglion cell types, small ganglion cells, and two types of displaced ganglion cell. The major retinal projection is contralateral, but a small, ipsilateral component also exists. Afferents from neurons which are postsynaptic to the thalamic retinal terminal fields also reach nBOR. Four distinct cell types were identified within the terminal field of nBOR: stellate neurons (63%), amacrine cells (19%), elongate neurons (14%), and large ganglionic neurons (4%). Both stellate and amacrine cells appear to be intrinsic neurons, while elongate and ganglionic neurons constitute the efferent neuron population of nBOR. In addition, cells which lie medial to the terminal field, pyriform and commissural neurons, send dendrites into nBOR. Pyriform neurons project to the nucleus of the medial longitudinal fasciculus (nMLF) and cranial nerve nuclei III and IV, while commissural neurons project to the contralateral nBOR. Large reticular neurons of the nMLF also send dendrites into nBOR. Efferent projections from nBOR were observed in the large-celled pretectal nucleus and in nucleus lateralis profundus. A second major projection originates from the peri-nBOR region and is associated with the oculomotor system and with the nMLF. Efferent projections from the nMLF to the vestibular nuclei and to the rostral spinal cord were also observed, as well as projections which reach the brainstem from the large-celled pretectal nucleus, the posterior thalamic and anterior mesencephalic central gray.